Photodynamic therapy has been used as a method for the eradication of neoplastic cells from autologous grafts for cancer treatments. This method relies on the use of photosensitizing dyes, which when activated with light of a particular wavelength, produce toxic O2 radicals, ultimately leading to cell death. Photochemical treatments have also been used for pathogen inactivation, such as in “decontamination” of blood and blood-derived products. The danger of pathogen transmission through transfusion of whole blood, platelets concentrates, plasma and/or red blood cells still represent major concerns in medicine. Although there has been impressive progress in the prevention and maintenance of blood safety regarding the presence of microorganisms, blood components continue to carry risk of pathogen transfusion. Moreover, the presence of viruses in blood components is also of great concerns, mainly for the presence of Hepatitis C and human immunodeficiency virus (HIV), even though the risk of contamination is reduced to negligible levels. The presence of other viruses is also required and includes the human T-cell lymphotrophic virus type 1 (HTLV-1), Hepatitis B (HBV) and cytomegalovirus. Photodynamic compounds such as pseuralens, porphyrines, riboflavines and dimethyl of methylene bleue have been used in the treatment of pathogen in blood product. These compounds necessitate radiation by a ultra violet A lamp (UVA) to get activated, thus leading to possible mutagenic effect in the remaining cells present in the treated samples. (Corash, L., Inactivation of infectious pathogens in labile blood components: meeting the challenge, Transfus Clin Biol, 2001, 8, 138-145 Lin, L., Londe, H., Janda, M. J., Hanson, C. V. and Corash, L., Photochemical inactivation of pathogenic bacteria in human platelet concentrates, Blood, 1994, 83, 9, 2698-2706; Lin, L, Londe, H., Hanson, C. V., Wiesehahn, G., Isaacs, S., Cimino, G. and Corash, L., Photochemical inactivation of cell-associated human immunodeficiency virus in platelet concentrates, Blood, 1993, 82, 1, 292-297; Lin, L., Eiesehahn, G. P., Morel, P. A. and Corash, L., Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates, Blood, 1989, 74, 1, 517-525; Lin, L., Cook, D. N., Wiesehahn, G. P., Alfonso, R., Behrman, B., Cimino, G. D., Corten, L., Damonte, P. B., Dikeman, R., Dupuis, K., Fang. Y. M., Hanson, C. V., Heasrt, J. E., Lin, C. Y., Londe, H., Metchette, K., Nerio, A. T., Pu, J. T., Reames, A. A., Rheinschmidt, M., Tessman, J., Isaacs, S. T., Wollowitz, S. and Corash, L., Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light, Transfusion, 1997, 37, 423-435). Because of the UVA exposure to blood components, these techniques are not entirely satisfactory. There was therefore a need for new light sensitive derivatives that do not necessitate UVA exposure of blood components and that may also be a safer ad more acceptable replacement to UVA treated blood components.
Immunologic disorders are uncontrolled cell proliferations that result from the production of immune cells recognizing normal cells and tissues as foreign. After a variable latency period during which they are clinically silent, cells with immunoreactivity towards normal cells induce damages in these normal cells and tissues. Such immunologic disorders are usually divided in alloimmune conditions and autoimmune conditions. Alloimmune disorders occur primarily in the context of allogeneic transplantation (bone marrow and other organs: kidney, heart, liver, lung, etc.). In the setting of bone marrow transplantation, donor immune cells present in the hematopoietic stem cell graft react towards host normal tissues, causing graft-versus-host disease (GVHD). The GVHD induces damage primarily to the liver, skin, colon, lung, eyes and mouth. Autoimmune disorders are comprised of a number of arthritic conditions, such as rhumatoid arthritis, scleroderma and lupus erythematosus; endocrine conditions, such as diabetes mellitus; neurologic conditions, such as multiple sclerosis and myasthenia gravis; hematological disorders, such as autoimmune hemolytic anemia, etc. The immune reaction, in both alloimmune and autoimmune disorders, progresses to generate organ dysfunction and damage.
Despite important advances in treatment, immunologic complications remain the primary cause of failure of allogeneic transplantations, whether in hematopoietic stem cell transplantation (GVHD) or in solid organ transplantation (graft rejection). In addition, autoimmune disorders represent a major cause of both morbidity and mortality. Prevention and treatment of these immune disorders has relied mainly on the use of immunosuppressive agents, monoclonal antibody-based therapies, radiation therapy, and more recently molecular inhibitors. Significant improvement in outcome has occurred with the continued development of combined modalities, but for a small number of disorders and patients. However, for the most frequent types of transplantations (bone marrow, kidney, liver, heart and lung), and for most immune disorders (rheumatoid arthritis, connective tissue diseases, multiple sclerosis, etc.) resolution of the immunologic dysfunction and cure has not been achieved. Therefore, the development of new approaches for the prevention and treatment of patients with immunologic disorders is critically needed particularly for those patients who are at high risk or whose disease has progressed and are refractory to standard immunosuppressive therapy. Allogeneic stem cell transplantation (AlloSCT) has been employed for the treatment of a number of malignant and non-malignant conditions. Allogeneic stem cell transplantation is based on the administration of high-dose chemotherapy with or without total body irradiation to eliminate malignant cells, and host hematopoietic cells. Normal hematopoietic donor stem cells are then infused into the patient in order to replace the host hematopoietic system. AlloSCT has been shown to induce increased response rates when compared with standard therapeutic options. One important issue that needs to be stressed when using AlloSCT relates to the risk of reinfusing immune cells that will subsequently recognize patient cells as foreign and cause GVHD. A variety of techniques have been developed that can deplete up to 105 of T cells from the marrow or peripheral blood. These techniques, including immunologic and pharmacologic purging, are not entirely satisfactory. One major consideration when purging stem cell grafts is to preserve the non-host reactive T cells so that they can exert anti-infectious and anti-leukemia activity upon grafting. The potential of photodynamic therapy, in association with photosensitizing molecules capable of destroying immunologically reactive cells while sparing normal host-non-reactive immune cells, to purge hematopoietic cell grafts in preparation for AlloSCT or autologous stem cell transplantation (AutoSct), and after AlloSCT in the context of donor lymphocyte infusions to eliminate recurring leukemia cells has largely been unexplored. To achieve eradication of T cells, several approaches have been proposed including:                1) in vitro exposure of the graft to monoclonal antibodies and immunotoxins against antigens present on the surface of T cells (anti-CD3, anti-CD6, anti-CD8, etc.);        2) in vitro selection by soybean agglutinin and sheep red blood cell rosetting;        3) positive selection of CD34+ stem cells; and        4) in vivo therapy with combinations of anti-thymocyte globulin, or monoclonal antibodies.        5) In vitro exposure of recipient-reactive donor T cells by monoclonal antibodies or immunotoxins targeting the interleukin 2 receptor or OX-40 antigen (Cavazzana-Calvo M. et al. (1990) Transplantation, 50:1-7; Tittle T. V. et al (1997) Blood 89:4652-58; Harris D. T. et al. (1999) Bone Marrow Transplantation 23:137-44).        
However, most of these methods are not specifically directed at the alloreactive T cell subset and associated with numerous problems, including disease recurrence, graft rejection, second malignancies and severe infections. In addition, the clinical relevance of several of these methods remains to be established.
There are many reports on the use of photodynamic therapy in the treatment of malignancies (Daniell M. D., Hill J. S. (1991) Aust. N. Z J Surg., 61: 340-348). The method has been applied for cancers of various origins and more recently for the eradication of viruses and pathogens (Raab O. (1990) Infusoria Z. Biol., 39:524).
The initial experiments on the use of photodynamic therapy for cancer treatment using various naturally occurring or synthetically produced photoactivable substances were published early this century (Jesionek A., Tappeiner V. H. (1903) Muench Med Wochneshr, 47: 2042; Hausman W. (1911) Biochem. Z., 30: 276). In the 40's and 60's, a variety of tumor types were subjected to photodynamic therapy both in vitro and in vivo (Kessel, David (1990) Photodynamic Therapy of neoplastic disease, Vol. I, II, CRC Press. David Kessel, Ed. ISBN 0-8493-5816-7 (v. 1), ISBN 0-8493-5817-5 (v. 2)). Dougherty et al. and others, in the 70's and 80's, systematically explored the potential of oncologic application of photodynamic therapy (Dougherty T. J. (1974) J Natl Cancer Inst., 51: 1333-1336; Dougherty T. J. et al. (1975) J. Natl Cancer Inst., 55: 115-121; Dougherty T. J. et al. (1978) Cancer Res., 38: 2628-2635; Dougherty T. J. (1984) Urol. Suppl., 23: 61; Dougherty T. J. (1987) Photochem. Photobiol., 45: 874-889).
Treatment of Immunoreactive Cells with Photodynamic Therapy
There is currently a lack of agents which allow selective destruction of immunoreactive cells while leaving intact the normal but suppressed residual cellular population. Preferential uptake of photosensitive dye and cytotoxicity of photodynamic therapy against leukemia (Jamieson C. H. et al. (1990) Leuk. Res., 14: 209-219) and lymphoid cells (Greinix H. T., et al. Blood (1998) 92:3098-3104; are reviewed in Zic J. A. et al. Therapeutic Apheresis (1999) 3:50-62) have been previously demonstrated.
It would be highly desirable to be provided with photosensitizers which possess at least one of the following characteristics:                i) preferential localization and uptake by the immunoreactive cells;        ii) upon application of appropriate light intensities, killing those cells which have accumulated and retained the photosensiting agents;        iii) sparing of the normal hemopoietic stem cell compartment from the destructive effects of activated photosensitizers; and        iv) potential utilization of photosensitizers for hematopoietic stem cell purging of immunoreactive cells, in preparation for allogeneic or autologous stem cell transplantation.        v) Potential utilization of photosensitizers for ex vivo elimination of reactive immune cells in patients with immunological disorders.The Rhodamine Dyes        
Rhodamine 123 (2-(6-amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester hydrochloride), a lipophilic cationic dye of the pyrylium class which can disrupt cellular homeostasis and be cytostatic or cytotoxic upon high concentration exposure and/or photodynamic therapy, although with a very poor quantum yield (Darzynkiewicz Z., Carter S. (1988) Cancer Res., 48: 1295-1299). It has been used in vitro as a specific fluorescent stain for living mitochondria. It is taken up and is preferentially retained by many tumor cell types, impairing their proliferation and survival by altering membrane and mitochondrial function (Oseroff A. R. (1992) In Photodynamic therapy (Henderson B. W., Dougherty T. J., eds) New York: Marcel Dekker, pp. 79-91). In vivo, chemotherapy with rhodamine 123 can prolong the survival of cancerous mice, but, despite initial attempts to utilize rhodamine 123 in the treatment of tumors, the systemic toxicity of rhodamine 123 may limit the usefulness (Bernal, S. D., et al. (1983) Science, 222: 169; Powers, S. K. et al. (1987) J. Neurosur., 67: 889).
U.S. Pat. No. 4,612,007 issued on Sep. 16, 1986 in the name of Richard L. Edelson, discloses a method for externally treating human blood, with the objective of reducing the functioning lymphocyte population in the blood system of a human subject. The blood, withdrawn from the subject, is passed through an ultraviolet radiation field in the presence of a dissolved photoactive agent capable of forming photoadducts with lymphocytic-DNA. This method presents the following disadvantages and deficiencies. The procedure described is based on the utilization of known commercially available photoactive chemical agents for externally treating patient's blood, leaving the bone marrow and potential resident leukemic clones intact in the process. According to Richard L. Edelson, the method only reduces, does not eradicate, the target cell population. Moreover, the wavelength range of UV radiation used in the process proposed by Richard L. Edelson could be damageable to the normal cells.
International Application published on Jan. 7, 1993 under International publication number WO 93/00005, discloses a method for inactivating pathogens in a body fluid while minimizing the adverse effects caused by the photosensitive agents. This method essentially consists of treating the cells in the presence of a photoactive agent under conditions that effect the destruction of the pathogen, and of preventing the treated cells from contacting additional extracellular protein for a predetermined period of time. This method is concerned with the eradication of infectious agents from collected blood and its components, prior to storage or transfusion.
It would be highly desirable to be provided with new rhodamine derivatives for the treatment of immunereactive cells which overcomes these drawbacks while having no systemic toxicity for the patient.
Halogenated rhodamine salts are dyes that have the property of penetrating cells and generally localising at the mitochondria. They have been used in conjunction with photoactivation to kill certain types of cells, namely cancer cells in Leukemia, and activated T-cells in autoimmune diseases.
The generally accepted mechanism for the cell killing effect is the production of singlet oxygen which is the reactive intermediate in the disruption of the life-sustaining biological processes of the cell.
The role of the rhodamine dye in the production of singlet oxygen is that of a photosensitizer, i.e. that of a molecule which absorbs the incident light energy and transfers it to ground state oxygen, thereby elevating it to its singlet excited state which is the reactive intermediate.
It is further known that the efficiency of the energy transfer process is greatly enhanced by the presence of heavy atoms such as halogens on the aromatic chromophore of the dye.
One critical problem that has not been addressed however is the differential uptake of the photosensitizer by the target cells relative to the other, normal, cells. Indeed, it is known that uptake is generally a function of the molecular structure of the dye being absorbed and that this property varies with different cell types.
It would therefore be highly desirable to be provided with a series of new halogenated rhodamine dyes bearing a variety of substituents at different positions of the molecule thereby making available new selective dyes for specific target cells.
One aim of the present invention is to produce new photosensitizers endowed with the following characteristics:                i) preferential localization and uptake by the immunoreactive cells;        ii) upon application of appropriate light intensities, killing those cells which have accumulated and retained the photosensiting agents;        iii) sparing of the normal hemopoietic stem cell compartment from the destructive effects of activated photosensitizers;        iv) potential utilization of photosensitizers for hematopoietic stem cell purging of immunoreactive cells in preparation for allogeneic or autologous stem cell transplantation; and        v) Potential utilization of photosensitizers for ex vivo elimination of reactive immune cells in patients with immunological disorders.        
Therefore, in accordance with the present invention, there is provided a series of new rhodamine derivatives alone or in association with a pharmaceutically acceptable carrier; whereby photoactivation of said derivatives induces cell killing while unactivated derivatives of general structure represented by the formula (I), and salts thereof, are substantially non-toxic to cells.
In accordance with the present invention, there is also provided with the use of the photoactivable rhodamine derivatives according to the invention for the photodynamic treatment for the selective destruction and/or inactivation of immunologically reactive cells without affecting the normal cells and without causing systemic toxicity for the patient, wherein appropriate intracellular levels of said derivatives are achieved and irradiation of a suitable wavelength and intensity is applied.
In accordance with the present invention, there is also provided a method of prevention of graft-versus-host disease associated with allogeneic stem cell transplantation in a patient, which comprises the steps of:                a) activating lymphocytes from a donor by mixing donor cells with host cells for a time sufficient for a period of time sufficient for an immune reaction to occur,        b) substantially eliminating the activated lymphocytes of step a) with photodynamic therapy using a therapeutic amount of a photoactivable derivative or composition of claim 1 under irradiation of a suitable wavelength; and        c) performing allogenic stem cell transplantation using the treated mix of step b).        
In accordance with the present invention, there is provided a method for the treatment of immunologic disorder in a patient, which comprises the steps of:                a) harvesting said patient's hematopoietic cells;        b) ex vivo treating of the hematopoietic cells of step a) by photodynamic therapy using a therapeutic amount of a photoactivable derivative or composition of claim 1 under irradiation of a suitable wavelength; and        c) performing graft infusion or autograft transplantation using the treated hematopoietic cells of step b).        
The immunologic disorder may be selected from the group consisting of conditions in which self cells or donor cells react against host tissues or foreign targets, such as graft-versus-host disease, graft rejection, autoimmune disorders and T-cell mediated immunoallergies.
The hematopoietic cells may be selected from the group consisting of bone marrow, peripheral blood, and cord blood mononuclear cells.
For the purpose of the present invention the following terms are defined below.
The term “immunoreactive disorders” is intended to mean any alloimmune or autoimmune reaction and/or disorders.
In accordance with other aspects of the present invention, these rhodamine compounds which are prepared following the general strategy of halogenating known and readily available rhodamine dyes thereby generating a first and varied series of intermediates, which themselves can serve as potential photosensitizers or, use these halogenated rhodamines as intermediates in the synthesis of a second series of rhodamine dyes whereby one or more halogen has been substituted for one of the groups of structure (I). In the case where all of the halogens are replaced by new groups, a subsequent halogenation step is added to the sequence to obtain the desired compound of structure I (see FIGS. 1 to 5).
Testing of these compounds on various types of cells surprisingly revealed some of the candidate molecules to be non-toxic, more efficient and more selective than the known halogenated rhodamine dyes.